Journal: Matrix Biology Plus

Article Title: Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca 2+ dynamics

doi: 10.1016/j.mbplus.2024.100148

Figure Lengend Snippet: Ca 2+ analysis of mouse islets with changes in RTG stiffness. Representative traces of intracellular Ca 2+ as measured by fluorescence intensity in 5 individual cells in the same islet over time in islets (A) unencapsulated, or encapsulated in (B) 2.5 wt% RTG, (C) 5 wt% RTG, and (D) 10 wt% RTG. Bars indicate a 900 % change in Ca 2+ compared with the baseline Ca 2+ levels. (E) Representative image of a mouse islet encapsulated in 10 wt% RTG treated with 2 mM glucose. The yellow outlined cells were oscillating at low glucose and are numbered according to corresponding Ca 2+ trace in D. Representative traces of intracellular Ca 2+ as measured by fluorescence intensity in 5 individual cells in the same islet treated with the Piezo1 inhibitor GsMTx-4 (2.5 μM) over time in islets (F) unencapsulated, or encapsulated in (G) 2.5 wt% RTG, (H) 5 wt% RTG, and (I) 10 wt% RTG. Bars indicate a 900 % change in Ca 2+ compared with the baseline Ca 2+ levels. (J) Area fraction of active cells at 2 mM glucose to total islet area quantified in unencapsulated islets or islets encapsulated in 2.5 wt%, 5 wt%, or 10 wt% RTG treated with the GsMTx-4 inhibitor (2.5 μM) or the control (n = 3). (K) Area under the curve (AUC) of the 1st phase 11 mM glucose oscillation in unencapsulated islets or islets encapsulated in 2.5 wt%, 5 wt%, or 10 wt% RTG treated with the GsMTx-4 inhibitor (2.5 μM) or the control. Each data point represents a single islet (n = 4–8 islets, n = 3 mice). (L) Ca 2+ oscillation frequency during 11 mM glucose stimulation in unencapsulated islets or islets encapsulated in 2.5 wt%, 5 wt%, or 10 wt% RTG treated with the GsMTx-4 inhibitor (2.5 μM) or the control. Each data point represents a single islet (n = 6–12 islets, n = 3 mice). (M) Amplitude of Ca 2+ oscillations in unencapsulated islets or islets encapsulated in 2.5 wt%, 5 wt%, or 10 wt% RTG treated with the GsMTx-4 inhibitor (2.5 μM) or the control. Each data point represents a single islet (n = 6–12 islets, n = 3 mice). Error bars represent the mean +/- SEM. p < 0.05 indicates statistical significance as determine by ANOVA with Tukey’s post hoc analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D.

Techniques: Fluorescence, Control

Journal: Matrix Biology Plus

Article Title: Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca 2+ dynamics

doi: 10.1016/j.mbplus.2024.100148

Figure Lengend Snippet: Analysis of the RTG as a scaffold for islet culture and mechanotransduction interaction through focal adhesion formation. (A) Representative confocal microscopy images of mouse islet viability at 48hr where red represents dead cells stained with propidium iodide and green represents viable cells stained with fluorescein diacetate. (B) Viability data for mouse islets encapsulated in 2.5 wt%, 5 wt%, and 10 wt% RTG versus unencapsulated for 24 and 48-hr periods (n = 3). (C) Representative confocal microscopy images of the unoxidized (488 nm) and oxidized (405 nm) channels of the ROS sensor. (D) Analysis of reactive oxygen species using an adenoviral ROS sensor in 5 wt% and 10 wt% RTG encapsulated and unencapsulated mouse islets (n = 3). Data is presented as fold change in roGFP2 fluorescence intensity, calculated by dividing the fluorescence intensity of the oxidized state by the fluorescence intensity of the unoxidized state. Both channels were corrected to background prior to ratiometric calculation. (E) Representative western blot of Rho in whole mouse islets unencapsulated and encapsulated in 5 wt% and 10 wt% RTG treated with 11 mM glucose for 1hr (n = 4). GTPδS and GDP are the positive and negative controls, respectively. (F) Western blot quantification for Rho expression in mouse islets. Rho expression was normalized to total protein content via BCA assay. Error bars represent the mean +/- SEM. p < 0.05 indicates statistical significance as determine by ANOVA with Tukey’s post hoc analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D.

Techniques: Confocal Microscopy, Staining, Fluorescence, Western Blot, Expressing, BIA-KA

Journal: Matrix Biology Plus

Article Title: Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca 2+ dynamics

doi: 10.1016/j.mbplus.2024.100148

Figure Lengend Snippet: Analysis of mouse and human islet function with changes in RTG stiffness. (A) Secreted insulin normalized to insulin content for mouse islets encapsulated in 2.5 wt%, 5 wt% and 10 wt% RTG or unencapsulated islets at non-stimulatory (2 mM) and stimulatory (20 mM) glucose concentrations (n = 4). (B) The stimulation index, or the ratio of insulin secretion at 20 mM glucose to 2 mM glucose, of the same treatment groups in A (n = 4). (C) Secreted insulin normalized to insulin content for human islets encapsulated in 2.5 wt% and 10 wt% RTG or unencapsulated islets at non-stimulatory (2 mM) and stimulatory (20 mM) glucose concentrations (n = 5). Error bars represent the mean +/- SEM. p < 0.05 indicates statistical significance as determine by ANOVA with Tukey’s post hoc analysis.

Article Snippet: To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D.

Techniques:

Journal: Matrix Biology Plus

Article Title: Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca 2+ dynamics

doi: 10.1016/j.mbplus.2024.100148

Figure Lengend Snippet: Piezo1 localization and islet function analysis in mouse islets unencapsulated and encapsulated in RTG. (A) Representative western blot of Piezo1 in whole mouse islets unencapsulated and encapsulated in 2.5 wt%, 5 wt%, and 10 wt% RTG treated with 2 mM and 20 mM glucose for 1hr (n = 4). (B) Western blot quantification for Piezo1 expression in mouse islets. Piezo1 expression was normalized to total protein content via Ponceau staining and BCA assay. (C) GSIS with non-stimulatory (2 mM) and stimulatory (20 mM) glucose with (+) or without (-) the peptide inhibitor GsMTx-4 (2.5 μM) added to the glucose solution in 2.5 wt% and 10 wt% encapsulated or unencapsulated mouse islets (n = 4). (D) Stimulation index from 2 mM to 20 mM glucose with the GsMTx-4 inhibitor or the control as calculated from data in B (n = 4). (E) PFK activity, reported as milliunit/mL = nmole/min/mL, in 5 wt% or 10 wt% encapsulated or unencapsulated mouse islets treated with 11 mM glucose with the GsMTx-4 inhibitor or the control (n = 6). Data is normalized to the NoRTG control (no inhibition) condition. Error bars represent the mean +/- SEM. p < 0.05 indicates statistical significance as determine by ANOVA with Tukey’s post hoc analysis.

Article Snippet: To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D.

Techniques: Western Blot, Expressing, Staining, BIA-KA, Control, Activity Assay, Inhibition